2 Susceptibility of surfactants towards pancreatic digestion
2.2 Materials and Methods
2.2.1 Nonionic surfactants
Cremophor® EL (Polyoxyethylene glyceroltriricinoleat 35, Ph. Eur.; Polyoxyl 35 Castor Oil, USP/NF, BASF)
The main component of Cremophor® EL is glycerol-polyethylene glycol ricinoleate, which, together with fatty acid esters of PEG, represents the hydrophobic part of the product. The smaller, hydrophilic part consists of PEG and ethoxylated glycerol (Figure 13). Cremophor® EL is produced by ethoxylation of castor oil with 35 moles of ethylene oxide. The hydrophilic-lipophilic balance (HLB) lies between 12 and 14. The critical micelle concentration (CMC) is approximately 0.02%163. Cremophor® EL can be classified as type IIIA according to Small’s lipid classification system.
Cremophor® EL was developed by BASF for oral, topical and injectable formulations.
Drawbacks of the product are its bitter taste and histamine-related toxicological side effects (anaphylactic reactions, drop of blood pressure, haemolytic reactions) after injection163,164. Cremophor® EL is one of the components of the Gengraf® (Cyclosporin A) microemulsion, the Norvir® (Ritonavir) oral solution and soft gelatine capsule, and the Kaletra® (Ritonavir and Lopinavir) soft gelatine capsules51.
In 2012, BASF has expanded its dermatology and solubilisation range, and developed a new naming system for its products. The new Kolliphor® product family is now including the whole solubilisers range. The new trade name for Cremophor® EL is Kolliphor® EL. Nevertheless, the old trade names were used within this work.
Susceptibility of surfactants towards pancreatic digestion
CH2
CH2 CH2 CH2 CH CH2 CH3
O CH2 CH2 O H
C O
n
n
Cremophor EL R1,2,3=
Cremophor RH 40 R1,2,3=
7 5
n
CH CH2 CH3
O CH2 CH2 O H CH2
C
O 5
n 10
CH2 CH2 O H O
H
n n
H2C CH H2C O O
O R1 R2
R3 H2C
CH H2C
O CH2 CH2 O R6 O CH2 CH2 O
R5
O CH2 CH2 O R4
R4,5,6= H
CH2
H2C CH2 CH2 CH CH2 CH3
7 OH
n
H2C CH2 O H
CH2
CH2 CH2 CH2 CH CH2 CH3
O H C
O 7 5
CH CH2 CH3 OH
CH2 C
O 10 5
n
H2C CH2 O H
H
CH CH2 CH3 OH
H2C
5 10
R4,5,6=
n
C
R7 O CH2 CH2 O H
O n
C
R7 O
O
CH2 CH2 O C R7 O
R4,5,6,7=
R4,5,6,7= R1,2,3=
R1,2,3=
R1,2,3=
R1,2,3=
Figure 13 Chemical structures of the main constituents of Cremophor® EL and Cremophor® RH 40. The products consist of a complex mixture of polyethylene glycols, polyethoxylated glycerols, polyethoxylated fatty acids, and mono-, di-, and tri-esters of glycerol that are polyethoxylated to different degrees.
Susceptibility of surfactants towards pancreatic digestion Cremophor® RH 40 (macrogol glycerol hydroxystearate, Ph. Eur.; polyoxyl 40 hydrogenated castor oil, USP/NF, BASF)
The main constituent of Cremophor® RH 40 is glycerol polyethylene glycol oxystearate, which, together with fatty acid glycerol polyglycol esters, forms the hydrophobic part of the product (75-83%). The hydrophilic part consists of polyethylene glycols and glycerol ethoxylate (17-25%, Figure 13). Cremophor® RH 40 is produced by ethoxylation of hydrogenated castor oil with 40 moles ethylene oxide. The HLB value lies between 14 and 16.
Cremophor® RH 40 was developed after Cremophor® EL, to produce a solubiliser for oral use that was not bitter164,165. Cremophor® RH 40 is one of the components of the Kaletra® (Lopinavir and Ritonavir) oral solution, and the Neoral® (Cyclosporin A) oral solution and soft gelatin capsule51. The new trade name for Cremophor® RH 40 is Kolliphor® RH40.
Solutol® HS 15 (macrogol 15 hydroxystearate, Ph. Eur., polyoxyl 15 hydroxystearate, USP;
BASF)
Solutol® is a mixture of mono- and diesters of 12-hydroxy stearic acid and polyethylenglycol (Figure 14). Furthermore, about 30% free PEG is present in the mixture. Solutol® is produced by the reaction of 15 moles ethylene oxide per mole of 12-hydroxystearic acid. The hydrophilic-lipophilic balance lies between 14 and 16. According to the product information, the critical micelle concentration (CMC) lies between 0.005 and 0.02%. Gonzáles et al.
reported an CMC of 0.021% at 37°C144. Solutol® was developed in 1995 by BASF as a low toxicity solubiliser for injection solutions but has also gained interest in the formulation of oral lipid dosage forms34,130,144,164,166-168. The new trade name for Solutol® HS 15 is Kolliphor® HS 15.
n 10 5
10
O CH2 CH2
H O C CH CH2 CH3
OH CH2
O
n
O CH2 CH2
H OH
5
10 5
n
O CH2 CH2
H O C
O
CH2
CH2 HC CH2 CH3 OH
C O
CH CH2 CH3 O
Figure 14 Chemical structures of the constituents of Solutol® HS 15. The content of free macrogol is about 30%, about 65-70% are mono- and diesters of 12-hydroxy stearic acid and polyethylenglycol.
Susceptibility of surfactants towards pancreatic digestion
Labrafil® M1944 (oleoyl macrogolglycerides, Ph. Eur., oleoyl polyoxylglycerides USP/NF, Gattefossé)
Labrafil® M1944CS is obtained by the partial pegylation of apricot kernel oil and consists of mono-, di-, and triglycerides as well as mono- and di-fatty acid esters of PEG-300 (Figure 15). The dominating fatty acid fractions are oleic acid and linoleic acid. Labrafil® M1944CS is able to self-emulsify on contact with aqueous media forming a coarse dispersion. The HLB value is 4. Labrafil® M1944CS is mainly used as vehicle for highly lipophilic APIs in order to increase their oral bioavailability169,170.
Labrafil® is a typical example of a multi-component mixture which hampers adequate classification of the excipient into Small’s LCS. However, Müllertz et al.161 recently recommended to classify such mixtures according to the main component and intended use.
According to this guidance, Labrafil® can be classified as a polar lipid (class II).
H2C CH2 CH2 CH2 CH3
n
C
R O CH2 CH2 O H
O n
H2C CH3 H2C
HC H2C O
OH OH
C R O
C H2C
HC H2C O
O OH
C R
O O
R
Labrafil M 1944 CS R=
Gelucire 50/13 R=
7 7
16
H2C CH
H2C O C R C
R O
O
O
O C R
O
C O O
CH2 CH2 O C R O R
Figure 15 Chemical structures of Labrafil® M 1944CS and Gelucire® 50/13. Both excipients are mixtures of mono- di and triglycerides as well as mono and diesters of fatty acids and PEG.
Gelucire® 50/13 (stearoyl macrogolglycerides, Ph. Eur., stearoyl polyoxylglycerides USP/NF, Gattefossé)
Gelucire® 50/13 is a semi-solid mixture of mono- di and triglycerides (aprox. 20%) as well as mono and diesters of fatty acids and PEG 1500 (approx. 72%). In addition it contains about 8% free PEG (Figure 15). More than 90% of the fatty acid fraction are derivatives of stearic
Susceptibility of surfactants towards pancreatic digestion Gelucire® 50/13 is obtained by partial alcoholysis of saturated oils using macrogol. It is able to self-emulsify on contact with aqueous media forming a fine dispersion. The HLB value is 13. This excipient is mainly used in formulations with focus on solubility and bioavailability enhancement170,171. Gelucire® 50/13 can be classified as type IIIA according to Small’s lipid classification system.
2.2.2 Dispersion experiments
Surfactant dispersions were prepared by weighing appropriate amounts of the surfactant in a glass vial and adding Sørensen’s phosphate buffer to obtain a 1% (w/v) solution. Agitation was achieved using a magnetic stirrer. Since most of the surfactants were solid or semi-solid at room temperature, the complete container was heated to 50-60 °C and molten samples were mixed properly prior to use. This step was further crucial to avoid inhomogeneity as all surfactants used in this study represent multi-component mixtures. All samples were allowed to disperse completely prior to analysis. The mean particle size of the dispersions was assessed using either photon correlation spectroscopy (PCS; Malvern HPPS, Malvern Instruments, UK) or laser diffraction measurements (LD, Malvern Mastersizer 2000, Malvern Instruments, UK).
2.2.3 Digestion experiments
An appropriate amount of surfactant was weighed into a reaction vessel. Sørensen’s phosphate buffer and mixed micelle pre-concentrate was added. The solutions were allowed to equilibrate at 37°C. Constant agitation was provided either by a magnetic stirrer or by utilisation of the end-over-end shaker. In all cases, formulations were completely dispersed prior to initiation of simulated digestion.
Before start of the analysis, sodium chloride and calcium chloride corresponding to 150 mM and 5 mM respectively were added and the pH value of the mixture was adapted to 6.8 using a 1 N NaOH solution. The simulated digestion was started by the addition of pancreatin powder equivalent to 450 units of lipase activity per ml.
Two different monitoring methodologies were applied:
a) pH-stat titration which non-selectively detects the drop of the pH caused by the liberation of free fatty acid
b) analysis of fatty acids derived from the specific surfactant by means of HPTLC coupled with in situ densitometric analysis
A detailed description of the methods is given in chapter III.
Susceptibility of surfactants towards pancreatic digestion
Estimation of the extent of surfactant digestion
The proportional extent of the digestion of simple components can be achieved by comparison of the mol of digestion products relative to the mol of ester bonds initially present and, therefore, potentially digestible. This can be easily achieved for well defined compounds such as triglycerides but is more challenging for multi-component mixtures. The analytical method which was used for the monitoring of the in vitro digestion experiments was not capable to analyse the starting material and the whole spectrum of potential digestion products. In order to estimate the mol of cleavable FA present in 1 g of the surfactant, the ester value of each surfactant was used.
The ester value IE is the number that expresses the quantity of potassium hydroxide in milligrams required to saponify the esters present in 1 g of the substance. It is calculated from the saponification value IS and the acid value IA according to the following equation:
IE = IS - IA eq. 3
The saponification value IS is the number that expresses the quantity of potassium hydroxide (KOH) in milligrams to saponify the esters and neutralise the free acids in 1 g of a sample.
The acid value IA is the number that expresses the quantity of potassium hydroxide in milligrams required to neutralise the free acids present in 1 g of the substance.